The present invention relates to a process for producing linear D-.beta.-hydroxyalkanoic acids, more particularly to a process for economically producing D-.beta.-hydroxyalkanoic acids from inexpensive alkanoic acids or 2-alkenoic acids by utilizing microorganisms having an ability capable of converting the alkanoic acids or 2-alkenoic acids stereospecifically into the D-forms of .beta.-hydroxyalkanoic acids.
D-.beta.-hydroxyalkanoic acids are useful substances as raw materials for synthesizing optically active medicines, agricultural chemicals, perfumes and the like. In particular, the D-.beta.-hydroxyalkanoic acid having 4 carbon atoms, i.e. D-.beta.-hydroxybutyric acid (hereinafter referred to as "D-.beta.-HBA"), that having 5 carbon atoms, i.e. D-.beta.-hydroxyvaleric acid (hereinafter referred to as "D-.beta.-HVA"), that having 6 carbon atoms, i.e. D-.beta.-hydroxycaproic acid (hereinafter referred to as "D-.beta.-HCA") and that having 7 carbon atoms, i.e. D-.beta.-hydroxyheptanoic acid (hereinafter referred to as "D-.beta.-HHA") are considered to be important.
With respect to processes for producing these D-.beta.-hydroxyalkanoic acids, no instance in which D-.beta.-HBA, D-.beta.-HVA, D-.beta.-HCA and D-.beta.-HHA are all prepared by the same process, is reported. Since processes for producing the respective compounds except for D-.beta.-HHA are reported, they will be explained below.
As to chemical processes, A. I. Meyers and Gerald Knaus succeeded in synthesizing D-.beta.-HHB or D-.beta.-HVA through chiral oxazolines as reported in Tetrahedron Letters, No. 14, 1333(1974). Also, a process for preparing ethyl eters of D-.beta.-HBA or D-.beta.-HVA from the corresponding .beta.-ketoesters using an asymmetrically modified nickel catalyst is reported by A. Tai in Yukagaku, 29(11), 44(1980). Further, synthesis of D-.beta.-HCA by enantioselective aldol condensation is reported by D. A. Evans, J. Bartroli and T. L. Shih in Journal of American Chemical Society, 103(8), 2127(1981). These chemical processes require the use of expensive chemicals, and accordingly are not suitable for the industrial production from the economical point of view.
On the other hand, the following processes are known as processes for producing D-forms of .beta.-hydroxyalkanoic acids using microorganisms.
(1) Fermentative method: There is reported by R. M. Lafferty, as disclosed in Japanese Unexamined Patent Publication No. 18794/1978, a method for producing D-.beta.-HBA by employing bacteria such as Alcaligenes eutrophus and Bacillus megaterium or their mutants unable to assimilate D-.beta.-HBA with carbohydrates such as glucose or alcohols such as methanol as a carbon source. In this method, D-.beta.-HBA is obtained as a by-product by utilizing the fact that gram positive bacteria accumulate poly-(D-.beta.-HBA) therein, and accordingly the yield is very low. Further, the substance produced by this method is only D-.beta.-HBA, unlike a method utilizing a .beta.-oxidation enzyme system of fatty acids as intended by the present inventors to disclose herein.
(2) Hydrogenation method of .beta.-ketoacids or .beta.ketoesters: There is reported a method for producing D-.beta.-HVA, D-.beta.-HCA or D-.beta.-hydroxycaprylic acid (C.sub.8) by stereoselective hydrogenation of .beta.-ketoacids or .beta.-ketoesters using Baker's yeast [R. U. Lemieux and J. Giguere, Canadian Journal of Chemistry, 29, 678(1951); Gyorgy Frater, Helvetica Chimica Acta, 62, 2829(1979)]. However, .beta.-HBA obtained by treating acetoacetic acid or ethyl acetoacetate in the same manner is the L-form. This method is not economical, since the raw materials are expensive and large amounts of Baker's yeast and sucrose are required.
(3) Microbial optical resolution method: D-.beta.-HBA is obtained by optical resolution of DL-.beta.-HBA using Penicillium glaucum, as reported by A. Mckenzie and A. Harden in Journal of Chemical Society, 83, 430(1903). This method is not only poor in optical purity, but also uneconomical for industrial production.
(4) .beta.-Hydroxylation method of alkanoic acids or 2-alkenoic acids: It is well known as a general knowledge that decomposition of fatty acids in organisms proceeds through .beta.-hydroxyacyl-CoA. On the basis of this knowledge, there has been made a study of production of .beta.-hydroxyalkanoic acids from alkanoic acids or 2-alkenoic acids using microorganisms. C. T. Goodhue and J. R. Schaeffer disclose in U.S. Pat. No. 3,553,081 the production of L-.beta.-hydroxyisobutyric acid from isobutyric acid using Pseudomonas putida. S. Tahara and J. Mizutani report the production of, from trans-2-alkenoic acids having 6 to 12 carbon atoms, the corresponding L-.beta.-hydroxyalkanoic acids having 6 to 12 carbon atoms using Mucor sp. [Agricultural Biological Chemistry, 42(4), 879(1978)]. This method is considered to be the most superior as an industrial method to the before-mentioned other methods in that the raw materials are inexpensive and the mass production is relatively easy. However, .beta.-hydroxyalkanoic acids obtained by this method are all the L-forms, and no method for producing the D-forms has been known. Thus, the present inventors searched for microorganisms capable of converting isobutyric acid or methacrylic acid into D-.beta.-hydroxyisobutyric acid, and consequentially found the presence of microorganisms capable of producing the D-form of .beta.-hydroxyisobutyric acid, unlike the conventional knowledge (U.S. Pat. No. 4,310,635). Further, on the basis of this finding, the present inventors carried forward a study and also found that Candida rugosa IFO 0750 which is a strain capable of producing D-.beta.-hydroxyisobutyric acid can produce and accumulate D-.beta.-HVA from valeric acid, as disclosed in Japanese Unexamined Patent Publication No. 65188/1982. However, the yield of D-.beta.-HVA is very low. Also, production of .beta.-hydroxyalkanoic acids from the corresponding alkanoic acids having 4, 6 or 7 carbon atoms is very trace. Although the presence of microorganisms capable of converting not only branched alkanoic acids such as isobutyric acid, but also linear alkanoic acids into D-forms of .beta.-hydroxyalkanoic acids was found for the first time by the present inventors, it has been difficult to produce linear .beta.-hydroxyalkanoic acids in high yields because they are easily metabolized by the microorganisms producing them.